PROJECT SUMMARY/ABSTRACT Hearing loss is the most common sensory deficit worldwide. Disabling hearing loss will affect an estimated 900 million individuals globally by 2050 at an annual cost of US$ 750 billion. There is compelling socioeconomic rationale to devise novel therapeutic strategies to treat hereditary and non-hereditary forms of inner ear disease. Mouse models of deafness and vestibular dysfunction are most commonly exploited to test gene and pharmacotherapeutics designed to rescue sensory function. A widespread experimental approach is to deliver genes or drugs to the functionally immature neonatal inner ears of mice that model human deafness and then assess structural and functional recovery at mature stages. This work takes advantage of the plasticity of the pre-hearing mammalian inner ear to accommodate microinjection of aqueous reagents without significantly affecting acquisition of auditory or vestibular function. However, a pressing need is to define experimental systems that model the responsivity of the adult inner ear to therapeutic genetic manipulation. The conceptual basis of this proposal is that delivery of functionally silent genetic constructs to the fetal inner ear will enable atraumatic activation in differentiated cell types of mature inner ear. We hypothesize that transuterine microinjection of Cre recombinase-responsive genetic elements into the otic vesicle of mice harboring tamoxifen- inducible alleles will permit control of the timing and cell type-specificity of therapeutic gene delivery without compromising inner ear structure or function. Our long term goal is to verify where in the inner ear and when specific genes must be modulated to restore or protect auditory function in models of hereditary and non- hereditary hearing loss. In Aim 1, we will atraumatically deliver a chemically inducible genetic switch flanking green fluorescent protein (GFP) to the fetal inner ear using a recombinant adeno-associated viral vector (rAAV) and then pharmacologically trigger expression in the adult inner ear. We hypothesize that GFP expression will be constrained to inner or outer hair cells, subsets of supporting cells in the organ of Corti, to vestibular supporting cells in the cristae and maculae, and spiral ganglion neurons as predicated by relevant Cre driver alleles. In Aim 2, we will deploy the genetic switch system to reprogram adult mouse supporting cells into hair cells by conditional expression of the Pou4f3, Gfi1, and Atoh1 transcription factors. We hypothesize that exogenous bioactive signals will be efficiently transmitted to supporting cells in the adult mouse inner ear. In Aim 3, we will use an inducible hybrid transcriptional activation system to reprogram supporting cells into hair cells. We hypothesize that forced transcriptional activation of endogenous Pou4f3, Gfi1, and Atoh1 in adult mouse supporting cells will induce a hair cell fate. Successful completion of our aims may establish a mouse model system that enables in vivo validation of druggable genetic targets that can preserve hearing and balance in the mature inner ear.